Use of protoporphyrinogen oxidase

Abstract

The present invention relates to the use of a protoporphyrinogen oxidase. A method for controlling weeds comprises applying a herbicide containing an effective dose of a PPO inhibitor to a field in which at least one transgenic plant is present, wherein the transgenic plant comprises, in the genome thereof, a polynucleotide sequence encoding protoporphyrinogen oxidase, and the transgenic plant has reduced plant damage and/or has an increased plant yield compared with other plants that do not have a polynucleotide sequence encoding protoporphyrinogen oxidase. The protoporphyrinogen oxidase PPO1-PPO14 of the present invention has a high tolerance to a PPO inhibitor herbicide. In addition, the plants containing the polynucleotide sequence encoding protoporphyrinogen oxidase have a strong tolerance to the PPO-inhibitor herbicide, and show high-resistance tolerance to almost all of 4-fold field concentration of oxyfluorfen, saflufenacil and flumioxazin and 2-fold field concentration of sulfentrazone. Therefore, the protoporphyrinogen oxidase has broad application prospects in plants.

1 . A method for controlling weeds, characterized by comprising applying a herbicide containing an effective dose of a PPO inhibitor to a field where at least one transgenic plant is present, wherein the transgenic plant comprises in its genome a polynucleotide sequence encoding a protoporphyrinogen oxidase, and the transgenic plant has a reduced plant damage and/or increased plant yield compared with other plants without the polynucleotide sequence encoding the protoporphyrinogen oxidase, wherein the protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;
 preferably, the protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   preferably, the protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   further preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NOs:1-14;   preferably, the transgenic plant comprises monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plant is oats, wheat, barley, millet, corn, sorghum,  Brachypodium distachyon , rice, tobacco, sunflower, alfalfa, soybean,  Cicer arietinum , peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or  Arabidopsis thaliana ; further preferably, the transgenic plant is a glyphosate-tolerant plant, and the weeds are glyphosate-resistant weeds;   preferably, the PPO-inhibitor herbicide comprises a PPO-inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;   further preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone, and/or flumioxazin.   
     
     
         2 . The method for controlling weeds according to  claim 1 , characterized in that the polynucleotide sequence of the protoporphyrinogen oxidase comprises:
 (a) a polynucleotide sequence encoding an amino acid sequence having at least 88% sequence identity to a sequence selected from SEQ ID NOs: 1-14, wherein the polynucleotide sequence does not include SEQ ID NOs: 15-28; or   (b) a polynucleotide sequence as shown in any one of SEQ ID NOs: 29-42 or SEQ ID NOs: 62-64.   
     
     
         3 . The method for controlling weeds according to  claim 1 , characterized in that the transgenic plant further comprises at least one second polynucleotide encoding a second herbicide-tolerant protein, which is different from the polynucleotide sequence encoding the protoporphyrinogen oxidase. 
     
     
         4 . The method for controlling weeds according to  claim 3 , characterized in that the second polynucleotide encodes a selectable marker protein, a protein with synthetic activity, a protein with degradation activity, a biotic stress-resistant protein, an abiotic stress-resistant protein, a male sterility protein, a protein that affects plant yield and/or a protein that affects plant quality. 
     
     
         5 . The method for controlling weeds according to  claim 4 , characterized in that the second polynucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxy phenyl pyruvate dioxygenase, acetolactate synthase, and/or cytochrome-like protein. 
     
     
         6 . The method for controlling weeds according to  claim 1 , characterized in that the herbicide containing an effective dose of a PPO inhibitor further includes a glyphosate herbicide, glufosinate herbicide, auxin-like herbicide, graminicide, pre-emergence selective herbicide and/or post-emergence selective herbicide. 
     
     
         7 . A planting combination for controlling the growth of weeds, comprising a PPO-inhibitor herbicide and at least one transgenic plant, wherein the herbicide containing an effective dose of a PPO inhibitor is applied to a field where the at least one transgenic plant is present, wherein the transgenic plant comprises in its genome a polynucleotide sequence encoding a protoporphyrinogen oxidase, and the transgenic plant has a reduced plant damage and/or increased plant yield compared with other plants without the polynucleotide sequence encoding the protoporphyrinogen oxidase; wherein the protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;
 preferably, the protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   preferably, the protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   further preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NOs:1-14;   preferably, the transgenic plant comprises monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plant is oats, wheat, barley, millet, corn, sorghum,  Brachypodium distachyon , rice, tobacco, sunflower, alfalfa, soybean,  Cicer arietinum , peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or  Arabidopsis thaliana ; further preferably, the transgenic plant is a glyphosate-tolerant plant, and the weeds are glyphosate-resistant weeds,   preferably, the PPO-inhibitor herbicide comprises a PPO-inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;   further preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone, and/or flumioxazin.   
     
     
         8 . The planting combination for controlling the growth of weeds according to  claim 7 , characterized in that the polynucleotide sequence of the protoporphyrinogen oxidase comprises:
 (a) a polynucleotide sequence encoding an amino acid sequence having at least 88% sequence identity to a sequence selected from SEQ ID NOs: 1-14, wherein the polynucleotide sequence does not include SEQ ID NOs: 15-28; or   (b) a polynucleotide sequence as shown in any one of SEQ ID NOs: 29-42 or SEQ ID NOs: 62-64.   
     
     
         9 . The planting combination for controlling the growth of weeds according to  claim 7 , characterized in that the transgenic plant further comprises at least one second polynucleotide encoding a second herbicide-tolerant protein, which is different from the polynucleotide sequence encoding the protoporphyrinogen oxidase. 
     
     
         10 . The planting combination for controlling the growth of weeds according to  claim 9 , characterized in that the second polynucleotide encodes a selectable marker protein, a protein with synthetic activity, a protein with degradation activity, a biotic stress-resistant protein, an abiotic stress-resistant protein, a male sterility protein, a protein that affects plant yield and/or a protein that affects plant quality. 
     
     
         11 . The planting combination for controlling the growth of weeds according to  claim 10 , characterized in that the second polynucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxy phenyl pyruvate dioxygenase, acetolactate synthase, and/or cytochrome-like protein. 
     
     
         12 . The planting combination for controlling the growth of weeds according to  claim 7 , characterized in that the herbicide containing an effective dose of a PPO inhibitor further includes a glyphosate herbicide, glufosinate herbicide, auxin-like herbicide, graminicide, pre-emergence selective herbicide and/or post-emergence selective herbicide. 
     
     
         13 . A method for generating a plant which is tolerant to a PPO-inhibitor herbicide, characterized in that the method comprises introducing a polynucleotide sequence encoding a protoporphyrinogen oxidase into the genome of the plant, and when the herbicide containing an effective dose of a PPO inhibitor is applied to a field where at least the plant is present, the plant has a reduced plant damage and/or increased plant yield compared with other plants without the polynucleotide sequence encoding the protoporphyrinogen oxidase, wherein the protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;
 preferably, the protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   preferably, the protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   further preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NOs:1-14;   preferably, the introduction method comprises genetic transformation, genome editing or gene mutation methods;   preferably, the plant comprises monocotyledonous plants and dicotyledonous plants; more preferably, the plant is oats, wheat, barley, millet, corn, sorghum,  Brachypodium distachyon , rice, tobacco, sunflower, alfalfa, soybean,  Cicer arietinum , peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or  Arabidopsis thaliana;      preferably, the PPO-inhibitor herbicide comprises a PPO-inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;   further preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone, and/or flumioxazin.   
     
     
         14 . A method for cultivating a plant which is tolerant to a PPO-inhibitor herbicide, characterized by comprising:
 planting at least one plant propagule, wherein the plant propagule comprises in its genome a polynucleotide encoding a protoporphyrinogen oxidase, wherein the protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   allowing the plant propagule to grow into a plant; and   applying the herbicide comprising an effective dose of a PPO inhibitor to a field comprising at least the plant, and harvesting the plant having a reduced plant damage and/or increased plant yield compared with other plants without the polynucleotide sequence encoding the protoporphyrinogen oxidase;   preferably, the protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   preferably, the protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   further preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NOs:1-14;   preferably, the plant comprises monocotyledonous plants and dicotyledonous plants; more preferably, the plant is oats, wheat, barley, millet, corn, sorghum,  Brachypodium distachyon , rice, tobacco, sunflower, alfalfa, soybean,  Cicer arietinum , peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or  Arabidopsis thaliana;      preferably, the PPO-inhibitor herbicide comprises a PPO-inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;   further preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone, and/or flumioxazin.   
     
     
         15 . A method for protecting a plant from damages caused by a PPO-inhibitor herbicide or for conferring tolerance to the PPO-inhibitor herbicide upon a plant, characterized by comprising applying the herbicide comprising an effective dose of a PPO inhibitor to a field where at least one transgenic plant is present, wherein the transgenic plant comprises in its genome a polynucleotide sequence encoding a protoporphyrinogen oxidase, and the transgenic plant has a reduced plant damage and/or increased plant yield compared with other plants without the polynucleotide sequence encoding the protoporphyrinogen oxidase, wherein the protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;
 preferably, the protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14; 
 preferably, the protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14; 
 more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14; 
 further preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NOs:1-14; 
 preferably, the transgenic plant comprises monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plant is oats, wheat, barley, millet, corn, sorghum,  Brachypodium distachyon , rice, tobacco, sunflower, alfalfa, soybean,  Cicer arietinum , peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or  Arabidopsis thaliana;    
 preferably, the PPO-inhibitor herbicide comprises a PPO-inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones; 
 further preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone, and/or flumioxazin. 
 
     
     
         16 . Use of a protoporphyrinogen oxidase for conferring tolerance to a PPO-inhibitor herbicide upon a plant, wherein the protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;
 preferably, the protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   preferably, the protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-14;   further preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NOs:1-14;   preferably, the use of the protoporphyrinogen oxidase for conferring tolerance to a PPO-inhibitor herbicide upon a plant comprises applying the herbicide containing an effective dose of a PPO inhibitor to a field where at least one transgenic plant is present, wherein the transgenic plant comprises in its genome a polynucleotide sequence encoding the protoporphyrinogen oxidase, and the transgenic plant has a reduced plant damage and/or increased plant yield compared with other plants without the polynucleotide sequence encoding the protoporphyrinogen oxidase;   preferably, the plant comprises monocotyledonous plants and dicotyledonous plants; more preferably, the plant is oats, wheat, barley, millet, corn, sorghum,  Brachypodium distachyon , rice, tobacco, sunflower, alfalfa, soybean,  Cicer arietinum , peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or  Arabidopsis thaliana;      preferably, the PPO-inhibitor herbicide comprises a PPO-inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;   further preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone, and/or flumioxazin.   
     
     
         17 . The use of a protoporphyrinogen oxidase for conferring tolerance to a PPO-inhibitor herbicide upon a plant according to  claim 16 , characterized in that the polynucleotide sequence of the protoporphyrinogen oxidase comprises:
 (a) a polynucleotide sequence encoding an amino acid sequence having at least 88% sequence identity to a sequence selected from SEQ ID NOs: 1-14, wherein the polynucleotide sequence does not include SEQ ID NOs: 15-28; or   (b) a polynucleotide sequence as shown in any one of SEQ ID NOs: 29-42 or SEQ ID NOs: 62-64.